Many cellular functions are carried out through large multi-protein complexes, rather than individual proteins. The systematic mapping of proteome-wide protein interactions to produce a comprehensive network of protein-protein interactions, or ‘interactome,’ is thus essential for understanding processes in biological systems. However, characterization of protein interactions is challenging because most protein interactions are transient, and many are unstable. In addition, multi-protein complexes possess no common factors or physical properties that can be used as an analytical handle.
Nonetheless, numerous efforts have been undertaken to profile large-scale protein interaction networks within a given cell, as the study of protein-protein interactions is a critical component in systems biology research directed at characterizing complex network interactions and behavior to better enable comprehension of protein function. Two popular methods to study protein-protein interactions are: (i) the yeast two-hybrid system, which is a genetic approach; and (ii) the use of protein cross-linking in combination with mass spectrometry (MS) to analyze purified protein complexes as well as their topological structures.
Protein cross-linking approaches can, at least in principal, not only identify what proteins interact within a complex, but can also pinpoint where proteins interact. However, despite much interest in profiling protein-protein interactions using cross-linking strategies and the availability of many commercial cross-linkers, (see, e.g., Pierce Chemicals, Double-Agents Cross-Linking Guide, 1999) few reports have illustrated progress in application/implementation of cross-linker approaches on a proteome-wide scale. This limitation is primarily due to inherent complexity in cross-linking reaction mixtures, which include large amounts of undesirable or unexpected products (see, e.g., Swaim, C. L. et al., J Am Soc Mass Spectrom 15:736-749, 2004), in addition to 1:1 linkage of interacting proteins. Moreover, the challenge of interpreting cross-linking approach results derives not only from the complexity of cross-linking reaction and digestion mixtures, but also from the complexity of MS/MS mass spectra—typically used for investigation of cross-linked peptides.
Therefore, there is a pronounced need in the art for novel cross-linking agents, and methods for using such agents, to simplify the identification and characterization of cross-linked proteins and peptides and thus facilitate the viability and utility of mass spectrometry for effective ‘interactome’ characterization.